Cancer predispositions in humans and severe DNA damage phenotypes in yeast result from defects in the Mre11-Rad50-Nbs1 (MRN) complex. MRN plays central and essential roles in repairing DNA double-strand breaks (DSBs) during homologous recombination repair as well as acting in meiosis, antibody hypermutation, telomere maintenance, and DNA damage signaling through ATM kinase. Yet, detailed mechanistic insights into these diverse MRN functions remain limited. The Mre11 nuclease complex with the Rad50 ATPase is conserved from archaea to humans and is regulated by Nbs1 in S. pombe and humans. We propose three Specific Aims to advance knowledge of MRN structural biochemistry, conformations, and interactions relevant to DNA damage repair and signaling functions. To accomplish these Aims, we will apply advanced biophysical techniques, including synchrotron solution X-ray scattering and atomic resolution crystal structure technologies in concert with genetic and mutational analyses in yeast. The proposed integrated biophysical and genetic studies will test hypotheses regarding Mre11's role in DNA target specificity and processing, Rad50's role in ATP-induced conformational controls and architectural interactions, and Nbs1's role in modulating Mre11 and Rad50 activities. The expected results will characterize functionally key Mre11, Rad50 and Nbs1 protein-protein and protein-DNA interfaces, conformations, and interaction architectures. Furthermore, the DNA damage sensitivity observed in the absence of any one member of the MRN complex suggests that our results will form a platform to test the utility of inhibitors that increase cellular sensitivity to ionizing radiation and other DNA damaging agents used for cancer radiotherapy and chemotherapy. Overall the results will connect MRN to cellular outcomes and human disease by defining interactions and mechanisms controlling genetic integrity, cancer resistance, radiotherapy resistance, and predispositions to cancer.